WL Nutrient Medium

Intended Use

Recommended for detection of motility and hydrogen sulphide production by pure cultures.

Composition**

Final pH (at 25°C): 7.3±0.2

**Formula adjusted, standardized to suit performance parameters

# Equivalent to Beef extract

Directions

Suspend 10.44 grams in 100 ml warm purified/distilled water. Heat to boiling with constant agitation to dissolve the medium completely. Dispense in tubes in 4 ml amounts and sterilize by autoclaving at 115°C (10 lbs pressure) for 15 minutes. Allow the tubed medium to cool in an upright position.

Principle And Interpretation

Motility Sulphide Medium was originally formulated by Edwards and Bruner (1) and further modified by Hajna (2) for the determination of motility and hydrogen sulphide production. The medium is also used for indirect evidence of motility by non-fermenting gram-negative bacilli. Proteose peptone and HM peptone B provide nitrogen compounds, carbon, sulphur and trace elements essential for bacterial growth. L-cystine and ferric ammonium citrate are the H2S indicators. Ferric ammonium citrate also provides extra nutrients for citrate-utilizing bacteria. Agar and gelatin preserve an intact stab line. Motile organisms grow away from stab line showing diffused growth while non-motile organisms grow along the stab line. Hydrogen sulphide production is indicated by the blackening of the medium. Due to the free L-cystine, generally negative organisms may give a positive reaction (3). After observing motility and H2S production, same medium can be utilized to detect urea hydrolysis. The culture in the medium is overlaid with 1 ml of Urea Broth (M111A) and incubated at 35°C for upto 6 hours. A urease positive reaction is observed as a reddish-purple colour formation in the Urea Broth.

Type of specimen

Isolated Microorganism

Specimen Collection and Handling:

With inoculating needle, stab centre of medium to approximately one-half of depth.(3)

After use, contaminated materials must be sterilized by autoclaving before discarding.

Warning and Precautions :

Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling specimens. Safety guidelines may be referred in individual safety data sheets.

Limitations

• 1. Growth from an 18-24 hr pure culture should be used. (3)
• 2. Motility Sulfide Medium permits H2S production from L-cystine. However, H2S reactions on this medium may differ from the reactions usually obtained by a group of organisms since it contains free L-cystine which may give a positive reaction by organisms considered negative by classical methods.(e.g. KIA/TSA).(3)

Performance and Evaluation

Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.

Quality Control

Appearance
Cream to yellow homogeneous coarse powder

Gelling
Semisolid, comparable with 0.4% Agar gel and 8.0% Gelatin gel.

Colour and Clarity of prepared medium
Yellow clear to slightly opalescent gel forms in tubes as butts

Reaction
Reaction of 10.44% w/v aqueous solution at 25°C. pH: 7.3±0.2

pH
7.10-7.50

Cultural Response
Cultural characteristics observed after an incubation at 35 - 37°C for 18 - 24 hours

Key: (*) Corresponding WDCM numbers. (#) Formerly known as Enterobacter aerogenes

Storage and Shelf Life

Store between 10-30°C in a tightly closed container and the prepared medium at 15-30°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition. Seal the container tightly after use. Product performance is best if used within stated expiry period.

Disposal

User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with sample must be decontaminated and disposed of in accordance with current laboratory techniques (4,5).

Reference

1. Edwards P. R. and Brunner D. W., 1942, Circulation of the Kentucky Agricultural Experimental Station, No. 54.
2. Hajna A. A., 1950, Public Health Lab., 8:36.
3. MacFaddin J. F., 1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol. 1, Williams and Wilkins, Baltimore.
4. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
5. Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.